We investigated venous sampling of the pituitary venous drainage to aid in the diagnosis and treatment of patients with Cushing's syndrome. Over 700 patients have now received bilateral simultaneous inferior petrosal sinus (IPS) sampling. The results indicate that 1) the procedure can be performed successfully in all patients with Cushing's syndrome (successful sampling has been performed in over 99% of the patients in whom it has been attempted); 2) the procedure distinguishes patients with ectopic ACTH secretion from those with pituitary adenomas with nearly 100% accuracy; 3) IPS sampling successfully determines in which side of the pituitary gland microadenomas reside in patients with Cushing's disease with 70% accuracy; and 4) unilateral inferior petrosal sinus sampling, which is commonly used clinically, is frequently misleading. Repeat transsphenoidal surgery is successful in elimubating the hypercortisolism of Cushing's disease in about 70% of patients. This therapy for patients with Cushing's disease after previous pituitary surgery had not previously been examined. Repeated sella exploration in the early postoperative period in patients who did not respond to the first operation was shown to be successful in most patients who received it. The subset of patients who are most likely to have success with early repeat surgery can be selected based on the findings during the first operation. MRI scanning with and without gadolinium-EDTA was used to evaluate patients with Cushing's disease preoperatively. This technique permitted identification of the adenoma in about 55% of those patients with surgically proven microadenomas. Proper timing of the MRI after administration of gadolinium-EDTA was critical in the optimal use of the technique. Pituitary adenomas were detected in 10% of 100 normal subjects with MRI scanning with contrast. The endocrine aberration in pituitary tumors and ectopic ACTH secreting tumors that cause Cushing's syndrome is loss of normal negative feedback regulation by cortisol. To investigate the basis of this, the structure of the pro-opiomelanocortin promoter region was investigated in pituitary and extrapituitary ACTH- producing tumors and demonstrated to be normal.